Pathological deposition of cholesterol underlies the development of vascular atherosclerotic lesions. The mechanism of cholesterol deposition is not known. This research project shows that platelets can mediate cholesterol accumulation within aortic smooth muscle cells. Washed platelets prepared from rat blood were incubated with cultured rat aortic smooth muscle cells in the presence or absence of trombin. All experiments were carried out in the absence of serum. Cholesterol accumulation within smooth muscle cells was detected histochemically using filipin staining. Cholesteryl ester lipid droplets accumulated in smooth muscle cells only when added platelets were activated by thrombin. Addition of increasing numbers of platelets resulted in increasing amounts of cholesteryl ester within cells. Cholesteryl ester accumulation occurred in confluent and non-confluent smooth muscle cell cultures. Addition of the cholesterol synthesis inhibitor mevinolin to smooth muscle cell cultures incubated with thrombin-activated platelets, did not diminish cholesteryl ester lipid droplet accumulation. Platelet-free supernatants were prepared from thrombin-activated platelets incubated separately from smooth muscle cells. When added to the cultured smooth muscle cells, the platelet-free supernatants also induced cholesteryl ester lipid droplet accumulation. These and other results indicate that activated-platelets secrete cholesterol that vascular-derived smooth muscle cells can accumulate. The significance of this research lies in the identification of a mechanism that can explain the pathologic accumulation of cholesterol in smooth muscle cells within atherosclerotic lesions.